Activation of naive CD4+ T cells (TCD4+) is driven by MHC class II (MHCII)-bound peptides (epitopes) displayed at the surfaces of dendritic cells (DCs) that have trafficked from sites of infection to regional lymph nodes. While most studies concentrate on the primary response, the majority of pathogen exposures in nature are repeat encounters that play out in decidedly different contexts. Indeed, we hypothesize that activation of memory TCD4+, increasingly appreciated as a key aspect of the recall response, is fundamentally distinct. This is attributable in large part to the presence of antigen-specific B cells, which possess unique antigen processing capabilities. Consequently, this drives a significant shift in the TCD4+ specificities that emerge from recall vs. primary responses. Through well-established influenza models that play to the experience of both co-PIs, this hypothesis will be pursued via two tightly linked specific aims. In the first aim the impact of B cells in the secondary response will be assessed by: a) selectively eliminating or turning on MHCII on memory B cells prior to a secondary challenge, and b) restricting flu protein expression to specific cell types by engineering viruses that contain selective miRNA targeting sequences. In the second aim the antigen processing and presenting capabilities of ex vivo flu-specific B cells vs. DCs will be assessed by in vitro TCD4+ activation assays. The prediction is that the observed differences between these antigen presenting cell (APC) types will provide a basis for the TCD4+ specificities that are amplified during the secondary vs. primary responses. This project is expected to set the stage for studies that explore the processing machinery that facilitates B cell-mediated presentation, the role of memory B cells in responses to other pathogens, as well as more applied studies that leverage resulting principles to enhance rational vaccine design.